Serologic correlates of protection against bacillus anthracis infection

ABSTRACT

Regions of  Bacillus anthracis  protective antigen are provided representing epitopes recognized by antibodies in subjects that have acquired immunity to  Bacillus anthracis  infection. The recognition of these epitopes correlates with autoimmunity in a subject. Also provided are vaccines that include at least one of these epitopes that when administered to a subject provide improved acquired immunity.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.13/577,878, filed Aug. 8, 2012, which is the U.S. National Phaseapplication of PCT/US2011/024317, filed Feb. 10, 2011, and claimspriority to U.S. Provisional Application No. 61/303,055 filed Feb. 10,2010, and U.S. Provisional Application No. 61/333,456 filed May 11,2010, the contents of each of which are incorporated herein byreference.

GOVERNMENT INTEREST

The invention described herein may be manufactured, used, and licensedby or for the United States Government.

FIELD OF THE INVENTION

The invention relates to physiologically relevant epitope sequencesrelated to acquired immunity to Bacillus anthracis protective antigen(PA). The peptide sequences of the invention represent previouslyunidentified regions of PA that elicit an immune response in a mammal.Particularly, the invention presents epitopes targeted by the immunesystem in Rhesus following vaccination with rPA or AVA.

BACKGROUND OF THE INVENTION

Anthrax is caused by infection with Bacillus anthracis, a spore-forming,rod-shaped bacterium. The dormant spore-form is highly resistant toextreme conditions, high temperatures, and a variety of chemicaltreatments. The spores gain entry either through an open wound causingcutaneous disease, by ingestion causing gastrointestinal disease, or areinhaled causing inhalation anthrax. All three forms can progress to asystemic infection leading to shock, respiratory failure, and death.(Mock, M. and Mignot, T, (2003) Cell Microbiol., 5(1):15-23). Thestability of the spores, and their infectious capacity, make them aconvenient bioterrorist weapon.

The two known toxins of B. anthracis are binary combinations ofprotective antigen (PA), named for its ability to induce protectiveimmunity against anthrax, with either edema factor (EF) or lethal factor(LF). PA is the cell biding component of both toxins and is responsiblefor bringing the catalytic EF or LF into the host cells. EF is anadenylate cyclase which converts ATP to cyclic AMP and causes edema(Brossier, F. & Mock, M, 2001, Toxicon. 39(11):1747-55). The combinationof PA-EF forms edema toxin (ETx) which causes edema when injectedlocally. LF is a zinc-dependent endoprotease known to target theamino-terminus of the mitogen-activated protein kinase kinase (MAPKK)family of response regulators (Id.). The cleavage of these proteinsdisrupts a signaling pathway and leads to cytokine dysregulation andimmune dysfunction. LF combined with PA forms lethal toxin (LTx) whichis lethal when injected on its own. It is also known that there arefatal anthrax cases where administration of antibiotics and clearance ofbacteria have failed to rescue the patient. This indicates that theremay be a “point of no return” level of LTx in the blood that may predictthe outcome of infection.

Development of a safe and effective vaccine for inhalation and otherforms of anthrax infection is vital to the health and safety of thepopulation and an essential component of any bioterrorism defensestrategy. Additionally, the identification of targeted therapiesfollowing anthrax infection is essential to managing a patientpopulation. As such, there exists a need for vaccines and treatments aswell as methods for determining whether post-vaccination protection isachieved prior to possible anthrax exposure and infection.

SUMMARY OF THE INVENTION

The following summary of the invention is provided to facilitate anunderstanding of some of the innovative features unique to the presentinvention and is not intended to be a full description. A fullappreciation of the various aspects of the invention can be gained bytaking the entire specification, claims, drawings, and abstract as awhole.

A process of determining protection against B. anthracis infection in asubject is provided that includes obtaining a biological sample from asubject, optionally after a first onset time, and screening thebiological sample for the presence or absence of antibodies to one ormore predefined regions of Bacillus anthracis protective antigen. Thepresence or absence of these antibodies allows one to determine thepresence or level of protection against B. anthracis. Some embodimentsinclude a prior administration of a Bacillus anthracis vaccine includingan immunogen corresponding to amino acid regions 181-210, 201-230,221-250, 241-270, 301-330, 321-350, 341-370, 361-390, 421-450, 561-590,or 581-610 of Bacillus anthracis protective antigen, a fragment thereof,or an analogue thereof, to the subject prior to obtaining the biologicalsample. A subject is optionally vaccinated with an AVA vaccine or arecombinant protective antigen vaccine.

The predefined region of Bacillus anthracis protective antigen isoptionally at least one of amino acid region 181-210, 201-230, 221-250,241-270, 301-330, 321-350, 341-370, 361-390, 421-450, 561-590, or581-610 of SEQ ID NO: 1.

A process optionally includes a second administering of the vaccine,obtaining a second biological sample following a second onset time, andscreening the second biological sample for the presence or absence ofantibodies to one or more predefined regions of Bacillus anthracisprotective antigen. The presence or level of protection against B.anthracis is then determined from the screening of the second biologicalsample.

Also provided is a process of eliciting an immune response in a subjectincluding administering a Bacillus anthracis vaccine including animmunogen corresponding to amino acid regions 181-210, 201-230, 221-250,241-270, 301-330, 321-350, 341-370, 361-390, 421-450, 561-590, or581-610 of SEQ ID NO: 1, a fragment thereof, or an analogue thereof, toa subject. A vaccine is optionally an isolated immunogen correspondingto amino acid regions 181-210, 201-230, 221-250, 241-270, 301-330,321-350, 341-370, 361-390, 421-450, 561-590, or 581-610 of SEQ ID NO: 1,a fragment thereof, or an analogue thereof. The immune response isoptionally the production of antibodies specific to Bacillus anthracisprotective antigen. The antibodies optionally neutralize lethal toxin.

Also provided is a vaccine that will produce acquired immunity toBacillus anthracis infection that includes an isolated immunogencorresponding to amino acid regions 181-210, 201-230, 221-250, 241-270,301-330, 321-350, 341-370, 361-390, 421-450, 561-590, or 581-610 of SEQID NO: 1, a fragment thereof, or an analogue thereof. A vaccineoptionally includes amino acid regions of Bacillus anthracis protectiveantigen.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents the reactivity of sera from AVA vaccinated Rhesusmacaques with peptides representing overlapping sequences of protectiveantigen;

FIG. 2 represents the reactivity of sera from AVA vaccinated rabbitswith peptides representing overlapping sequences of protective antigen;

FIG. 3 represents the reactivity of sera from AVA vaccinated humans withpeptides representing overlapping sequences of protective antigen;

FIG. 4 represents the regions of PA recognized by antibodies from Rhesusmacaque sera vaccinated with either AVA or rPA vaccines;

FIG. 5 represents the reactivity of sera from rPA vaccinated macaques atdifferent time points corresponding administration of 50 μg rPA with 14day interval where screening was done after administration of eachinjection starting from 3^(rd) dose till 7^(th) dose of rPA

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

The following description of particular embodiment(s) is merelyexemplary in nature and is in no way intended to limit the scope of theinvention, its application, or uses, which may, of course, vary. Theinvention is described with relation to the non-limiting definitions andterminology included herein. These definitions and terminology are notdesigned to function as a limitation on the scope or practice of theinvention but are presented for illustrative and descriptive purposesonly.

The invention has utility as a predictor of immunity to B. anthracisinfection. The invention has further utility as one or more peptidesequences that alone or when combined are improved vaccines conferringprotection against B. anthracis infection in a subject.

The invention provides polypeptide sequences that include relevantepitopes recognized by antibodies from subjects with acquired immunityto B. anthracis infection. The polypeptide sequences alone or incombination are useful for determining serologic correlates ofprotection to subsequent B. anthracis infection.

As such, a process for identifying or predicting immunity to infectionby B. anthracis is provided including screening for antibodies in asample obtained from a subject following vaccination with rPA, AVA, orfragments thereof, or prior infection by B. anthracis, to identifywhether antibodies to predefined regions of PA are generated by orpresent in the subject. The presence of antibodies to one or morepredefined regions of PA predicts the level of protection in a subjectagainst subsequent infection by B. anthracis. Standard vaccines for B.anthracis often require multiple administrations to produce the desiredlevel of immunity. Prior to the present invention, it was not possibleto determine whether a subject had acquired sufficient immunity afterone, two, three, or more administrations. The processes of the inventionprovide a mechanism by which a physician can identify whether aparticular subject needs additional vaccine administrations or hasalready developed the necessary protection against subsequent infectionby B. anthracis. The binding of antibodies from a subject to one or morepredefined regions of PA indicates the presence of acquired immunity.

As defined herein, a “predefined region” is a region of PA that servesas a B-cell epitope. A predefined region is a region of PA, optionallyhaving 30 amino acids or fewer, that is recognized by antibodies fromsubjects with immunity to B. anthracis infection. As such, the term“epitope” as used herein is synonymous with a predefined region. Apredefined region is optionally one or more of the following regions ofPA: the calcium ion chelating residues in the 1α₁ and 1β₁₃ strands,(AA181-210); 1β₁₃, 1α₂, 1β₁₄ (AA201-230); 1α₃ and 1α₄ (AA 221-250); and1α₄ and 2β₁ (AA241-270) regions in domain1; the chymotrypsin sensitiveloop 2β₂-2β₃ (AA 301-330); 2β₃ and 2α₁ (AA 321-350); 2α₁, 2β₄ and 2β₅(AA341-370); 2β₆ and 2β₇ (AA361-390); 2β₁₀, 2β₁₁, 2α₂ and 2β₁₂(AA421-450); 2β₁₃ in domain 2, 3α₃, 3α₄ (AA 561-590); and 3β₇, 3β₈(AA581-610) in domain 3 and partially in domain 4 of PA; fragmentsthereof; or combinations thereof.

A process optionally includes screening for antibodies to more than oneepitope. Screening is optionally performed following a singleadministration of vaccine. Optionally, screening is done after severalvaccinations. Optionally, screening is done after each of severalvaccinations. Illustratively, a subject is vaccinated with PA,recombinant PA, AVA, a vaccine as provided by the current invention,and/or other vaccine known in the art intended to provide immunityagainst B. anthracis infection, once, three times, five times, or moreand a biological sample such as blood is obtained from the subject fordetermination of the presence or absence of antibodies to predefinedregions of PA. Recognition of one or more antibodies to one or morepredefined regions of PA following vaccination correlates with apredicted level of protection to subsequent infection by B. anthracis.

As used herein, the term “anthrax” is intended to mean B. anthracis. Assuch, a subject suffering from anthrax is infected by B. anthracis.Similarly, anthrax such as virulent anthrax is the organism B.anthracis.

Interestingly, and in contrast to results expected from the prior art,the chymotrypsin sensitive loop 2β₂-2β₃ presents a strong epitope in PAthat is found following vaccination in humans, rabbits, and Rhesusmacaques. The 2β₂-2β₃ loop is involved in the transition of PA oligomersfrom prepore to pore. This region was not expected to show strongantigenicity in each of humans, rabbits, and Rhesus macaques, and tostrongly correlate with acquired immunity to virulent anthrax becauseeven though the structural region containing this loop may be importantimmunologically as a T cell epitope, recipients of vaccines such as AVAand rPA may not recognize this region as an antibody reactive B cellepitope. (Oscherwitz J, et al., Infect Immun, 2009; 77(8):3380-8. Assuch, this region of PA was not expected to be a good correlate ofimmunity. The presence of antibodies to this and surrounding regions ofPA in multiple species as identified in the present inventionsurprisingly demonstrates its importance as a correlate of immunity.

Antibody screening is accomplished by methods known in the art,illustratively, enzyme linked immunosorbent assay (ELISA), affinitychromatography, liquid chromatography, or other methods appreciated bythose of ordinary skill in the art. In some embodiments, a sample isobtained from a subject and screened in an ELISA assay using one or morepeptides representing epitopes in PA or non-epitope regions. A positiveresult is the presence of one or more antibodies in the sample to one ormore epitopes above background levels, optionally 2 times background,optionally 3 times background. In some embodiments an amino acidsequence from each of the four domains of PA, domain 1 (aa 1-258),domain 2 (aa 25-487), domain 3 (aa 488-495), and/or domain 4 (aa596-735) are represented by at least one peptide. It is appreciated thatthe numbering of predefined sequences represents the numbering of themature PA sequence. PA such as that illustrated in SEQ ID NO: 1, is freeof the putative signal sequence of 29 amino acids that is cleaved toproduce the mature PA protein. As such, the numbering presented hereinis related to mature PA.

The inventive epitopes are peptide regions of PA from B. anthracis (SEQID NO: 1).

(SEQ ID NO: 1) EVKQENRLLNE SESSSQGLLG YYFSDLNFQA PMVVTSSTTGDLSIPSSELE NIPSENQYFQ SAIWSGFIKV KKSDEYTFATSADNHVTMWV DDQEVINKAS NSNKIRLEKG RLYQIKIQYQRENPTEKGLD FKLYWTDSQN KKEVISSDNL QLPELKQKSSNSRKKRSTSA GPTVPDRDND GIPDSLEVEG YTVDVKNKRTFLSPWISNIH EKKGLTKYKS SPEKWSTASD PYSDFEKVTGRIDKNVSPEA RHPLVAAYPI VHVDMENIIL SKNEDQSTQNTDSQTRTISK NTSTSRTHTS EVHGNAEVHA SFFDIGGSVSAGFSNSNSST VAIDHSLSLA GERTWAETMG LNTADTARLNANIRYVNTGT APIYNVLPTT SLVLGKNQTL ATIKAKENQLSQILAPNNYY PSKNLAPIAL NAQDDFSSTP ITMNYNQFLELEKTKQLRLD TDQVYGNIAT YNFENGRVRV DTGSNWSEVLPQIQETTARI IFNGKDLNLV ERRIAAVNPS DPLETTKPDMTLKEALKIAF GFNESNGNLQ YQGKDITEFD FNFDQQTSQNIKNQLAELNV TNIYTVLDKI KLNAKMNILI RDKRFHYDRNNIAVGADESV VKEAHREVIN SSTEGLLLNI DKDIRKILSGYIVEIEDTEG LKEVINDRYD MLNISSLRQD GKTFIDFKKYNDKLPLYISN PNYKVNVYAV TKENTIINPS ENGDTSTNGI KKILIFSKKG YEIG

Illustratively, the epitope sequence for PA in the 2β₂2β₃ region isSEVHGNAEVHASFFDIGGSVSAGFSNSNSS (SEQ ID NO: 3) representing amino acids301-330 of SEQ ID NO: 1.

The terms “polypeptide,” “peptide,” are used interchangeably herein andare illustratively a chain of two or more amino acid residues. In someembodiments, a peptide suitable for use in the instant invention is theamino acid sequence for PA protein, fragments thereof, or analoguesthereof used alone or combined with other peptides or otherwiseimmunogenic sequence(s) or therapeutics. A peptide is optionally animmunogen. It is appreciated that an immunogen is any molecule used tovaccinate an organism. As such, an immunogen is optionally a peptide, anucleic acid, or combinations thereof.

As used herein a “subject” is a mammal. Optionally, a subject is a humanor non-human primate. Optionally, a subject is a dog, cat, equine,sheep, bovine, rabbit, pig, or murine.

As used herein, the term “biological sample” is defined as sampleobtained from a biological organism, a tissue, cell, cell culturemedium, or any medium suitable for mimicking biological conditions, orfrom the environment. Non-limiting examples include, saliva, gingivalsecretions, cerebrospinal fluid, gastrointestinal fluid, mucous,urogenital secretions, synovial fluid, blood, serum, plasma, urine,cystic fluid, lymph fluid, ascites, pleural effusion, interstitialfluid, intracellular fluid, ocular fluids, seminal fluid, mammarysecretions, vitreal fluid, nasal secretions, throat or nasal materials,and combinations thereof. It is appreciated that a biological sample isoptionally a cell, illustratively, cells of or related to the immunesystem. Cells illustratively include white blood cells. Illustrativeexamples of white blood cells include leukocytes such as T-cells,B-cells, and T-helper cells.

A biological sample is obtained from a subject by conventionaltechniques. For example, CSF is obtained by lumbar puncture. Blood isoptionally obtained by venipuncture, while plasma and serum areoptionally obtained by fractionating whole blood according to knownmethods.

In some embodiments, a vaccine is administered to a subject prior to,simultaneous with, or subsequent to obtaining a biological sample fromthe subject. Optionally, a vaccine is administered and then an onsettime elapses prior to obtaining a biological sample from the subject. Anonset time is a time that generally considered by those of skill in theart to be sufficient for a subject to produce an antibody to a portionof an immunogen, such as an immunogen of the prior art. Optionally, anonset time is 1, 2, 3, 4, 5, 6, or more days. Optionally, an onset timeis 1, 2, 3, 4, 5, 6, or more weeks. An onset time is optionally any timebetween 1 day and 60 days, or any fraction or specific time periodtherebetween.

A process optionally includes a first administration of a vaccine, afirst onset time, and then subsequently obtaining a first biologicalsample. A process optionally also includes determining whether a secondadministration is required by the presence or absence of an antibody toa peptide in the biological sample. Optionally, a second administrationis performed followed by a second onset time and obtaining a secondbiological sample for screening for the presence or absence ofantibodies to one or more peptides. This iterative process optionallycontinues until a subject demonstrates acquired immunity or a physiciandetermines that development of immunity is not possible in the subject.As such, a third, fourth, fifth, or additional administration isenvisioned under the invention. Similarly, a third, fourth, fifth, orsubsequent onset time is envisioned under the invention.

Also provided are vaccines that when administered to a subject willelicit an immune response. The term “immune response” refers to akinetic or magnitude variation of one or more elements of a subject'simmune system. An immune response is optionally the production ofantibodies that specifically recognize and interact with the vaccine.Non-limiting examples of immune responses include B-cell responses,calcium mobilization, calcium influx, or other changes in intracellularcalcium concentrations in any cellular compartment illustrativelyincluding the cytoplasm; nitric oxide production or release;phagocytosis; immunoglobulin uptake; production of immunoglobulin;alteration of protein phosphorylation; conversion of immune complexes;alteration of serum immunoglobulin levels; modulating the activity ofspleen tyrosine kinase (Syk), B-cell linker (BLNK), Burton's tyrosinekinase (Btk), Kit, Lck, Zap-70, Src, Stat1, SHP-2, phosphatidyl inositol3-kinase (PI3K), phosphoinositol 5-phosphatase, other kinases orphosphatases known in the art, phospholipase D, phospholipase C,sphingosine kinase; secretion of IL-1β, IL-6, IL-10, IL-2, IL-4, IFN-γ,Bcl10, TCR, TLR, or other cytokines, chemokines, or signaling molecules;interferon signaling; alteration of expression of interferon responsegene(s) (IRG); antibody production illustratively IgE or IgG production;alteration of the expression of any gene that encodes for a protein, aswell as the functional activity of any protein listed in Table 1;alteration of expression or activity of My4+/LeuM3− molecule; protectionfrom challenge after exposure to infectious organism; alteration innitrite levels; B-cell responses in various immune compartments;lymphoma cell responses; natural killer cell responses; monocyteresponses; macrophage responses; platelet responses; dendritic cellresponses; any immune cell response; Th1 and Th2 cytokine responses invarious immune compartments; immune cell maturation; activation orinhibition of an intracellular signaling pathway such as the NF-kappa Bsignaling pathway; apoptosis; alteration in allotype or isotype antibodylevels; in vitro recognition of antigen; survival; other response knownin the art; or combinations thereof.

It is appreciated that the peptides of the invention are representativevaccines operable under the invention. As such, any peptide vaccinedescribed herein is suitable in the inventive processes for determiningwhether a subject has acquired immunity or is at risk for subsequentinfection by B. anthracis.

A vaccine optionally includes one or more predefined regions of PA, oran analogue thereof. In some embodiments, a vaccine is a nucleic acidsequence that encodes a predefined region of PA such that when thenucleic acid sequence is administered to a subject, the predefinedpeptide sequence is expressed by the subject to act as an immunogen forthe generation of antibodies to the predefined sequence.

Optionally, inventive peptide sequences representing predefined regionsof PA useful as vaccines are: the calcium ion chelating residues in the1α₁ and 1β₁₃ strands, (AA181-210); 1β₁₃, 1α₂, 1β₁₄ (AA201-230); 1α₃ and1α₄ (AA 221-250); and 1α₄ and 2β₁ (AA241-270) regions in domain1; thechymotrypsin sensitive loop 2β₂-2β₃ (AA 301-330); 2β₃ and 2α₁ (AA321-350); 2α₁, 2β₄ and 2β₅ (AA341-370); 2β₆ and 2β₇ (AA361-390); 2β₁₀,2β₁₁, 2α₂ and 2β₁₂ (AA421-450); 2β₁₃ in domain 2, 3α₃, 3α₄ (AA 561-590);and 3β₇, 3β₈ (AA581-610) in domain 3 and partially in domain 4 of PA;analogues thereof, fragments thereof; or combinations thereof. When apeptide is used as a vaccine, analogues of a peptide are operable as animmunogen.

Optionally, peptides are recombinant and obtained by methods known inthe art. Illustratively, a nucleotide sequence is cloned into a plasmidwhich is transfected into E. coli and expressed. The nucleotide sequenceencoding immature PA is illustrated as SEQ ID NO: 2.

(SEQ ID NO: 2) AATTTCAATA TAATATAAAT TTAATTTTAT ACAAAAAGGAGAACGTATAT GAAAAAACGA AAAGTGTTAA TACCATTAATGGCATTGTCT ACGATATTAG TTTCAAGCAC AGGTAATTTAGAGGTGATTC AGGCAGAAGT TAAACAGGAG AACCGGTTATTAAATGAATC AGAATCAAGT TCCCAGGGGT TACTAGGATACTATTTTAGT GATTTGAATT TTCAAGCACC CATGGTGGTTACTTCTTCTA CTACAGGGGA TTTATCTATT CCTAGTTCTGAGTTAGAAAA TATTCCATCG GAAAACCAAT ATTTTCAATCTGCTATTTGG TCAGGATTTA TCAAAGTTAA GAAGAGTGATGAATATACAT TTGCTACTTC CGCTGATAAT CATGTAACAATGTGGGTAGA TGACCAAGAA GTGATTAATA AAGCTTCTAATTCTAACAAA ATCAGATTAG AAAAAGGAAG ATTATATCAAATAAAAATTC AATATCAACG AGAAAATCCT ACTGAAAAAGGATTGGATTT CAAGTTGTAC TGGACCGATT CTCAAAATAAAAAAGAAGTG ATTTCTAGTG ATAACTTACA ATTGCCAGAATTAAAACAAA AATCTTCGAA CTCAAGAAAA AAGCGAAGTACAAGTGCTGG ACCTACGGTT CCAGACCGTG ACAATGATGGAATCCCTGAT TCATTAGAGG TAGAAGGATA TACGGTTGATGTCAAAAATA AAAGAACTTT TCTTTCACCA TGGATTTCTAATATTCATGA AAAGAAAGGA TTAACCAAAT ATAAATCATCTCCTGAAAAA TGGAGCACGG CTTCTGATCC GTACAGTGATTTCGAAAAGG TTACAGGACG GATTGATAAG AATGTATCACCAGAGGCAAG ACACCCCCTT GTGGCAGCTT ATCCGATTGTACATGTAGAT ATGGAGAATA TTATTCTCTCAAAAAATGAGGATCAATCCA CACAGAATAC TGATAGTCAAACGAGAACAA TAAGTAAAAA TACTTCTACA AGTAGGACACATACTAGTGA AGTACATGGA AATGCAGAAG TGCATGCGTCGTTCTTTGAT ATTGGTGGGA GTGTATCTGC AGGATTTAGTAATTCGAATT CAAGTACGGT CGCAATTGAT CATTCACTATCTCTAGCAGG GGAAAGAACT TGGGCTGAAA CAATGGGTTTAAATACCGCT GATACAGCAA GATTAAATGC CAATATTAGATATGTAAATA CTGGGACGGC TCCAATCTAC AACGTGTTACCAACGACTTC GTTAGTGTTA GGAAAAAATC AAACACTCGCGACAATTAAA GCTAAGGAAA ACCAATTAAG TCAAATACTTGCACCTAATA ATTATTATCC TTCTAAAAAC TTGGCGCCAATCGCATTAAA TGCACAAGAC GATTTCAGTT CTACTCCAATTACAATGAAT TACAATCAAT TTCTTGAGTT AGAAAAAACGAAACAATTAA GATTAGATAC GGATCAAGTA TATGGGAATATAGCAACATA CAATTTTGAA AATGGAAGAG TGAGGGTGGATACAGGCTCG AACTGGAGTG AAGTGTTACC GCAAATTCAAGAAACAACTG CACGTATCAT TTTTAATGGA AAAGATTTAAATCTGGTAGA AAGGCGGATA GCGGCGGTTA ATCCTAGTGATCCATTAGAA ACGACTAAAC CGGATATGAC ATTAAAAGAAGCCCTTAAAA TAGCATTTGG ATTTAACGAA TCGAATGGAAACTTACAATA TCAAGGGAAA GACATAACCG AATTTGATTTTAATTTCGAT CAACAAACAT CTCAAAATAT CAAGAATCAGTTAGCGGAAT TAAACGTAAC TAACATATAT ACTGTATTAGATAAAATCAA ATTAAATGCA AAAATGAATA TTTTAATAAGAGATAAACGT TTTCATTATG ATAGAAATAA CATAGCAGTTGGGGCGGATG AGTCAGTAGT TAAGGAGGCT CATAGAGAAGTAATTAATTC GTCAACAGAG GGATTATTGT TAAATATTGATAAGGATATA AGAAAAATAT TATCAGGTTA TATTGTAGAAATTGAAGATA CTGAAGGGCT TAAAGAAGTT ATAAATGACAGATATGATAT GTTGAATATT TCTAGTTTAC GGCAAGATGGAAAAACATTT ATAGATTTTA AAAAATATAA TGATAAATTACCGTTATATA TAAGTAATCC CAATTATAAG GTAAATGTATATGCTGTTAC TAAAGAAAAC ACTATTATTA ATCCTAGTGAGAATGGGGAT ACTAGTACCA ACGGGATCAA GAAAATTTTAATCTTTTCTA AAAAAGGCTA TGAGATAGGA TAAGGTAATT CTAGGTGATT TTTAAATTA

It is appreciated that any portion of SEQ ID NO: 2, or a variantthereof, that will encode a peptide of the invention is operable hereinfor the production of a peptide or for use as a vaccine itself. Aninventive nucleic acid sequence that encodes a peptide of SEQ ID NO: 1,SEQ ID NO: 3, other peptide sequences herein, fragments thereof, oranalogues thereof is encompassed in the invention. Similarly, variantsof SEQ ID NO: 2, or fragments thereof are operable to encode a peptideof the invention. The genetic code is a degenerate code whereby specificnucleic acid sequences encode for particular amino acids, yet in mostcases more than one codon (three nucleotide sequence) will encode forthe same amino acid. It is therefore appreciated that a variant of SEQID NO: 2, or a fragment thereof, that encodes a polypeptide under theinvention is a nucleic acid sequence of the invention. It is well withinthe level of those of skill in the art to determine a nucleic acidsequence that will encode the inventive peptide immunogens.

A vaccine according to the invention includes a predefined sequence ofPA or an analogue thereof. Amino acids present in a vaccine or peptideinclude the common amino acids alanine, cysteine, aspartic acid,glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine,leucine, methionine, asparagine, proline, glutamine, arginine, serine,threonine, valine, tryptophan, and tyrosine as well as less commonnaturally occurring amino acids, modified amino acids or syntheticcompounds, such as alpha-asparagine, 2-aminobutanoic acid or2-aminobutyric acid, 4-aminobutyric acid, 2-aminocapric acid(2-aminodecanoic acid), 6-aminocaproic acid, alpha-glutamine,2-aminoheptanoic acid, 6-aminohexanoic acid, alpha-aminoisobutyric acid(2-aminoalanine), 3-aminoisobutyric acid, beta-alanine,allo-hydroxylysine, allo-isoleucine, 4-amino-7-methylheptanoic acid,4-amino-5-phenylpentanoic acid, 2-aminopimelic acid,gamma-amino-beta-hydroxybenzenepentanoic acid, 2-aminosuberic acid,2-carboxyazetidine, beta-alanine, beta-aspartic acid, biphenylalanine,3,6-diaminohexanoic acid, butanoic acid, cyclobutyl alanine,cyclohexylalanine, cyclohexylglycine, N5-aminocarbonylornithine,cyclopentyl alanine, cyclopropyl alanine, 3-sulfoalanine,2,4-diaminobutanoic acid, diaminopropionic acid, 2,4-diaminobutyricacid, diphenyl alanine, N,N-dimethylglycine, diaminopimelic acid,2,3-diaminopropanoic acid, S-ethylthiocysteine, N-ethylasparagine,N-ethylglycine, 4-aza-phenylalanine, 4-fluoro-phenylalanine,gamma-glutamic acid, gamma-carboxyglutamic acid, hydroxyacetic acid,pyroglutamic acid, homoarginine, homocysteic acid, homocysteine,homohistidine, 2-hydroxyisovaleric acid, homophenylalanine, homoleucine,homoproline, homoserine, homoserine, 2-hydroxypentanoic acid,5-hydroxylysine, 4-hydroxyproline, 2-carboxyoctahydroindole,3-carboxyisoquinoline, isovaline, 2-hydroxypropanoic acid (lactic acid),mercaptoacetic acid, mercaptobutanoic acid, sarcosine,4-methyl-3-hydroxyproline, mercaptopropanoic acid, norleucine, nipecoticacid, nortyrosine, norvaline, omega-amino acid, ornithine, penicillamine(3-mercaptovaline), 2-phenylglycine, 2-carboxypiperidine, sarcosine(N-methylglycine), 2-amino-3-(4-sulfophenyl)propionic acid,1-amino-1-carboxycyclopentane, 3-thienylalanine,epsilon-N-trimethyllysine, 3-thiazolylalanine, thiazolidine 4-carboxylicacid, alpha-amino-2,4-dioxopyrimidinepropanoic acid, and2-naphthylalanine. A peptide optionally has between 2 and about 60 aminoacids.

A peptide is obtained by any of various methods known in the artillustratively including isolation from a cell or organism, chemicalsynthesis, expression of a nucleic acid sequence, and partial hydrolysisof proteins. Chemical methods of peptide synthesis are known in the artand include solid phase peptide synthesis and solution phase peptidesynthesis or by the method of Hackeng, T M, et al., Proc Natl Acad SciUSA, 1997; 94(15):7845-50 or those reviewed by Miranda, L P, PeptideScience, 2000, 55:217-26 and Kochendoerfer G G, Curr Opin Drug DiscovDevel. 2001; 4(2):205-14. In some embodiments, the polypeptide sequencesare chemically synthesized by Fmoc synthesis.

The present invention encompasses an isolated peptide derived fromBacillus anthracis. An inventive PA immunogen has the sequencerepresented by a fragment of SEQ ID NO: 1 wherein the fragment includesat least a portion of: the calcium ion chelating residues in the 1α₁ and1β₁₃ strands, (AA181-210); 1β₁₃, 1α₂, 1β₁₄ (AA201-230); 1α₃ and 1α₄ (AA221-250); and 1α₄ and 2β₁ (AA241-270) regions in domain1; thechymotrypsin sensitive loop 2β₂-2β₃ (AA 301-330); 2β₃ and 2α₁ (AA321-350); 2α₁, 2β₄ and 2β₅ (AA341-370); 2β₆ and 2β₇ (AA361-390); 2β₁₀,2β₁₁, 2α₂ and 2β₁₂ (AA421-450); 2β₁₃ in domain 2, 3α₃, 3α₄ (AA 561-590);3β₇, 3β₈ (AA581-610) in domain 3 and partially in domain 4 of PA; orcombinations thereof. A peptide immunogen is optionally recombinant.However, it is also envisioned that naturally occurring PA immunogen maybe isolated from at least a portion of the cellular and other samplematerial for which the wild-type sequence is normally found. Methods forpurification of protein from organism derived samples are known and arewithin the level of skill in the art, illustratively affinitychromatography.

To ease purification procedures, the expressed polypeptides optionallyinclude a tag sequence. Illustrative examples of tags suitable for usein the instant invention include poly-histidine, CBP, CYD (covalent yetdissociable NorpD peptide), strep-2, FLAG, HPC or heavy chain of proteinC peptide tag, or GST and MBP protein fusion tag systems. It isappreciated that other tag systems are similarly operable. In someembodiments, recombinant peptides are expressed in E. coli and purifiedusing an affinity tag system followed by enzymatic cleavage of the tagsuch as by incorporating a factor Xa, thrombin, or other enzyme cleavagesite in the expressed polypeptide. Methods of tag cleavage are known inthe art and any effective method is appreciated to be suitable for usein the instant invention.

It is recognized that numerous analogues of a peptide are within thescope of the present invention including amino acid substitutions,alterations, modifications, or other amino acid changes that increase,decrease, or do not alter the function or immunogenic propensity of theinventive immunogen. Several post-translational modifications aresimilarly envisioned as within the scope of the present inventionillustratively including incorporation of a non-naturally occurringamino acid(s), phosphorylation, glycosylation, sulfation, and additionof pendent groups such as biotynlation, fluorophores, lumiphores,radioactive groups, antigens, or other molecules.

It is appreciated that the inventive peptides of the present inventionare phosphorylated or unphosphorylated. Optionally, an inventive peptideis disulfide bonded. Disulfide bonds can be to amino acid residueswithin the sequence or to a second polypeptide or molecule.

Modifications and changes can be made in the structure of the inventivepeptides that are the subject of the application and still obtain amolecule having similar or improved characteristics as the wild-typesequence (e.g., a conservative amino acid substitution). For example,certain amino acids can be substituted for other amino acids in asequence without appreciable loss of immunogenic activity. Because it isthe interactive capacity and nature of a polypeptide that defines thatpolypeptide's biological functional activity, certain amino acidsequence substitutions can be made in a polypeptide sequence andnevertheless obtain a polypeptide with like or improved properties.Optionally, a polypeptide is used that has less or more immunogenicactivity compared to the wild-type sequence.

In making such changes, the hydropathic index of amino acids can beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a polypeptide is generallyunderstood in the art. It is known that certain amino acids can besubstituted for other amino acids having a similar hydropathic index orscore and still result in a polypeptide with similar biologicalactivity. Each amino acid has been assigned a hydropathic index on thebasis of its hydrophobicity and charge characteristics. Those indicesare: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine(+2.8); cysteine/cysteine (+2.5); methionine (+1.9); alanine (+1.8);glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9);tyrosine (−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5);glutamine (−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9);and arginine (−4.5).

It is believed that the relative hydropathic character of the amino aciddetermines the secondary structure of the resultant polypeptide, whichin turn defines the interaction of the polypeptide with other molecules,such as enzymes, substrates, receptors, antibodies, antigens, and thelike. It is known in the art that an amino acid can be substituted byanother amino acid having a similar hydropathic index and still obtain afunctionally equivalent polypeptide. In making such changes, thesubstitution of amino acids whose hydropathic indices are within ±2 isoptional, those within ±1 are optional, and those within ±0.5 aresimilarly optional.

Substitution of like amino acids can also be made on the basis ofhydrophilicity, particularly, where the biological functional equivalentpolypeptide or peptide thereby created is intended for use inimmunological embodiments. The following hydrophilicity values have beenassigned to amino acid residues: arginine (+3.0); lysine (+3.0);aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine(+0.2); glutamine (+0.2); glycine (0); proline (−0.5±1); threonine(−0.4); alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine(−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine(−2.3); phenylalanine (−2.5); tryptophan (−3.4). It is understood thatan amino acid can be substituted for another having a similarhydrophilicity value and still obtain a biologically equivalent, and inparticular, an immunologically equivalent polypeptide. In such changes,the substitution of amino acids whose hydrophilicity values are within±2 is optional, those within ±1 are optional, and those within ±0.5 areoptional.

As outlined above, amino acid substitutions are generally based on therelative similarity of the amino acid side-chain substituents, forexample, their hydrophobicity, hydrophilicity, charge, size, and thelike. Exemplary substitutions that take various of the foregoingcharacteristics into consideration are well known to those of skill inthe art and include (original residue: exemplary substitution): (Ala:Gly, Ser), (Arg: Lys), (Asn: Gln, His), (Asp: Glu, Cys, Ser), (Gln:Asn), (Glu: Asp), (Gly: Ala), (His: Asn, Gln), (Ile: Leu, Val), (Leu:Ile, Val), (Lys: Arg), (Met: Leu, Tyr), (Ser: Thr), (Thr: Ser), (Tip:Tyr), (Tyr: Trp, Phe), and (Val: Ile, Leu). Embodiments of thisdisclosure thus contemplate functional or biological equivalents of apolypeptide as set forth above. In particular, embodiments of thepolypeptides can include variants having about 50%, 60%, 70%, 80%, 90%,and 95% sequence identity to the polypeptide of interest.

It is appreciated that amino acids are optionally L- or D-isomers. Aninventive polypeptide optionally includes mixtures of L- and D-isomers.

Peptide expression is illustratively accomplished from transcription ofa nucleic acid sequence encoding a peptide of the invention, andtranslation of RNA transcribed from nucleic acid sequence, modificationsthereof, or fragments thereof. Protein expression is optionallyperformed in a cell based system such as in E. coli, Hela cells, orChinese hamster ovary cells. It is appreciated that cell-free expressionsystems are similarly operable.

It is recognized that numerous analogues of a peptide are within thescope of the present invention including amino acid substitutions,alterations, modifications, or other amino acid changes that increase,decrease, or do not alter the function or the ability of PA immunogen togenerate antibodies that will interact with a wild-type PA proteinsequence. It is appreciated that an analogue includes one or more aminoacid insertions, deletions, substitutions, or modifications. An analogueof SEQ ID NO: 1, SEQ ID NO: 3, or any other amino acid sequence taughtherein is sufficiently immunogenic in a host to produce an antibody thatwill specifically bind to at least a portion of wild-type PA. One ofordinary skill in the art understands how to produce antibodies bystandard techniques and screen the resulting monoclonal or polyclonalantibodies for their ability to interact with an epitope sequence. Suchmethods are illustratively taught by Monoclonal Antibodies: Methods andProtocols, Albitar, M, ed., Humana Press, 2010 (ISBN 1617376469); andAntibodies: A Laboratory Manual, Harlos, E, and Lane, D. eds., ColdSpring Harbor Laboratory Press, 1988 (ISBN-10: 0879693142).

Further aspects of the present disclosure concern the purification, andin particular embodiments, the substantial purification, of a peptide.The term “purified” or “isolated” peptide as used herein, is intended torefer to a composition, isolatable from other components, wherein the PAimmunogen is purified to any degree relative to its naturally-obtainablestate. A purified peptide, therefore, also refers to a peptide free fromthe environment in which it may naturally occur.

Generally, “purified” or “isolated” will refer to a peptide compositionthat has been subjected to fractionation to remove various othercomponents, and which composition substantially retains its expressedbiological activity. Where the term “substantially” purified is used,this designation will refer to a composition in which the protein orpeptide forms the major component of the composition, such asconstituting about 50% or more of the proteins in the composition.

Various methods for quantifying the degree of purification of thepeptide known to those of skill in the art in light of the presentdisclosure as based on knowledge in the art. These include, for example,determining the specific activity of an active fraction, or assessingthe number of peptides within a fraction by SDS/PAGE analysis. Anillustrative method for assessing the purity of a fraction is tocalculate the specific activity of the fraction, to compare it to thespecific activity of the initial extract, and to thus calculate thedegree of purity, herein assessed by a “-fold purification number”. Theactual units used to represent the amount of activity will, of course,be dependent upon the particular assay technique chosen to follow thepurification and whether or not the expressed protein or peptideexhibits a detectable activity.

Various techniques suitable for use in peptide purification will be wellknown to those of skill in the art. These include, for example,precipitation with ammonium sulphate, polyethylene glycol, antibodiesand the like or by heat denaturation, followed by centrifugation;chromatography steps such as ion exchange, gel filtration, reversephase, hydroxylapatite and affinity chromatography; isoelectricfocusing; gel electrophoresis; and combinations of such and othertechniques. As is generally known in the art, it is believed that theorder of conducting the various purification steps may be changed, orthat certain steps may be omitted, and still result in a suitable methodfor the preparation of a substantially purified protein or peptide.

Additional methods of peptide isolation illustratively include columnchromatography, affinity chromatography, gel electrophoresis,filtration, or other methods known in the art. In some embodiments, animmunogen is expressed with a tag operable for affinity purification. Anillustrative tag is a 6× His tag. A 6× His tagged inventive peptideimmunogen is illustratively purified by Ni-NTA column chromatography orusing an anti-6× His tag antibody fused to a solid support. (GenewayBiotech, San Diego, Calif.) Other tags and purification systems aresimilarly operable.

It is appreciated that an inventive peptide is optionally not tagged. Inthis embodiment and other embodiments purification is optionallyachieved by methods known in the art illustratively includingion-exchange chromatography, affinity chromatography using antibodiesdirected to the peptide sequence of interest, precipitation with saltsuch as ammonium sulfate, streptomycin sulfate, or protamine sulfate,reverse phase chromatography, size exclusion chromatography such as gelexclusion chromatography, HPLC, immobilized metal chelatechromatography, or other methods known in the art. One of skill in theart may select the most appropriate isolation and purificationtechniques without departing from the scope of this invention.

There is no general requirement that the peptide always be provided inits most purified state. It is contemplated that less substantiallypurified products will have utility in certain embodiments. Partialpurification may be accomplished by using fewer purification steps incombination, or by utilizing different forms of the same generalpurification scheme. For example, it is appreciated that acation-exchange column chromatography performed utilizing an HPLCapparatus will generally result in a greater-fold purification than thesame technique utilizing a low pressure chromatography system. Methodsexhibiting a lower degree of relative purification may have advantagesin total recovery of protein product, or in maintaining the activity ofan expressed protein.

It is known that the migration of a peptide can vary, sometimessignificantly, with different conditions of SDS/PAGE (Capaldi et al.,Biochem. Biophys. Res. Comm., 76:425, 1977). It will, therefore, beappreciated that under differing electrophoresis conditions, theapparent molecular weights of purified or partially purified expressionproducts may vary.

PA immunogens of this invention may optionally be characterized byimmunological measurements including, without limitation, western blot,macromolecular mass determinations by biophysical determinations,SDS-PAGE/staining, HPLC and the like, antibody recognition assays, cellviability assays, apoptosis assays, and assays to infer immuneprotection or immune pathology by adoptive transfer of cells, proteinsor antibodies.

Also provided are isolated nucleic acids encoding the desired peptidesequence analogues thereof, or fragments thereof. These nucleic acidscan be used to produce the peptides of this invention or as nucleic acidvaccines, wherein the peptides of this invention are produced in asubject.

The term “nucleotide” is intended to mean a base-sugar-phosphatecombination either natural or synthetic, linear, circular and sequentialarrays of nucleotides and nucleosides, e.g. cDNA, genomic DNA, mRNA, andRNA, oligonucleotides, oligonucleosides, and derivatives thereof.Included in this definition are modified nucleotides which includeadditions to the sugar-phosphate groups as well as to the bases.

The term “nucleic acid” or “polynucleotide” refers to multiplenucleotides attached in the form of a single or double strandedpolynucleotide that can be natural, or derived synthetically,enzymatically, and by cloning methods. The term “oligonucleotide” refersto a polynucleotide of less than 200 nucleotides. The terms “nucleicacid” and “oligonucleotide” may be used interchangeably in thisapplication.

A nucleic acid as used herein refers to single- or double-strandedmolecules that may be DNA, including of the nucleotide bases A, T, C andG, or RNA, comprised of the bases A, U (substitutes for T), C, and G.The nucleic acid may represent a coding strand or its complement.Nucleic acids may be identical in sequence to the sequence naturallyoccurring, illustratively SEQ ID NO: 2 or a fragment thereof, or mayinclude alternative codons that encode the same amino acid as that foundin the naturally occurring sequence. Furthermore, nucleic acids mayinclude codons that represent conservative substitutions of amino acidsas are well known in the art.

The nucleic acid encoding the peptide of this invention can be part of arecombinant nucleic acid construct comprising any combination ofrestriction sites and/or functional elements as are well known in theart that facilitate molecular cloning and other recombinant DNAmanipulations. Thus, the present invention further provides arecombinant nucleic acid construct comprising a nucleic acid encoding apeptide of this invention.

The present invention also provides a vector with a nucleic acidsequence encoding an inventive PA immunogen sequence therein.Illustrative vectors include a plasmid, cosmid, cationic lipids,non-liposomal cationic vectors, cationic cyclodextrin, viruses with RNAor DNA genetic material, polyethylenimines, histidylated polylysine, orother vector system known in the art. A vector is optionally a plasmid.A suitable vector optionally possesses cell type specific expression orother regulatory sequences or sequences operable to stimulate or inhibitgene or protein expression. A vector illustratively contains a selectionmarker such as an antibiotic resistance gene.

The inventive nucleic acid sequence is optionally isolated from thecellular materials with which it is naturally associated. As usedherein, the term “isolated nucleic acid” means a nucleic acid separatedor substantially free from at least some of the other components of thenaturally occurring organism, for example, the cell structuralcomponents commonly found associated with nucleic acids in a cellularenvironment and/or other nucleic acids. The isolation of nucleic acidsis optionally accomplished by techniques such as cell lysis followed byphenol plus chloroform extraction, followed by ethanol precipitation ofthe nucleic acids. The nucleic acids of this invention can be isolatedfrom cells according to methods well known in the art for isolatingnucleic acids. Alternatively, the nucleic acids of the present inventioncan be synthesized according to standard protocols well described in theliterature for synthesizing nucleic acids. Modifications to the nucleicacids of the invention are also contemplated, provided that theessential structure and function of the peptide encoded by the nucleicacid are maintained.

Numerous methods are known in the art for the synthesis and productionof nucleic acid sequences illustratively including cloning andexpression in cells such as E. coli, insect cells such as Sf9 cells,yeast, and mammalian cell types such as Hela cells, Chinese hamsterovary cells, or other cells systems known in the art as amendable totransfection and nucleic acid and/or protein expression. Methods ofnucleic acid isolation are similarly recognized in the art.Illustratively, plasmid DNA amplified in E. coli is cleaved by suitablerestriction enzymes such as NdeI and XhoI to linearize PA DNA. The PADNA is subsequently isolated following gel electrophoresis using aS.N.A.P.™ UV-Free Gel Purification Kit (Invitrogen, Carlsbad, Calif.) asper the manufacturer's instructions.

Numerous agents are amenable to facilitate cell transfectionillustratively including synthetic or natural transfection agents suchas LIPOFECTIN, baculovirus, naked plasmid or other DNA, or other systemsknown in the art.

The nucleic acid sequences of the invention may be isolated or amplifiedby conventional uses of polymerase chain reaction or cloning techniquessuch as those described in conventional texts. For example, the nucleicacid sequences of this invention may be prepared or isolated from DNAusing DNA primers and PCR techniques. Alternatively, the inventive PAnucleic acid sequence may be obtained from gene banks derived fromBacillus anthracis whole genomic DNA. These sequences, fragmentsthereof, modifications thereto and the full-length sequences may beconstructed recombinantly using conventional genetic engineering orchemical synthesis techniques or PCR, and the like.

Also provided is a host cell transformed with an appropriate vector orwith the inventive PA peptide sequence. Illustrative host cells includeE. coli or Sf9 cells. Optionally, cell transfection is achieved byelectroporation.

Recombinant or non-recombinant proteinase peptides or recombinant ornon-recombinant proteinase inhibitor peptides or other non-peptideproteinase inhibitors can also be used in the present invention.Proteinase inhibitors are optionally modified to resist degradation, forexample degradation by digestive enzymes and conditions. Techniques forthe expression and purification of recombinant proteins are known in theart (see Sambrook Eds., Molecular Cloning: A Laboratory Manual 3^(rd)ed. (Cold Spring Harbor, N.Y. 2001).

Some embodiments of the present invention are compositions containing anucleic acid sequence that can be expressed as a peptide according tothe invention. The engineering of DNA segment(s) for expression in aprokaryotic or eukaryotic system may be performed by techniquesgenerally known to those of skill in recombinant expression. It isbelieved that virtually any expression system may be employed in theexpression of the claimed nucleic acid and amino acid sequences.

As used herein, the terms “engineered” and “recombinant” cells aresynonymous with “host” cells and are intended to refer to a cell intowhich an exogenous DNA segment or gene, such as a cDNA or gene has beenintroduced. Therefore, engineered cells are distinguishable fromnaturally occurring cells which do not contain a recombinantlyintroduced exogenous DNA segment or gene. A host cell is optionally anaturally occurring cell that is transformed with an exogenous DNAsegment or gene or a cell that is not modified. Engineered cells arecells having a gene or genes introduced through the hand of man.Recombinant cells include those having an introduced cDNA or genomicDNA, and also include genes positioned adjacent to a promoter notnaturally associated with the particular introduced gene.

To express a recombinant peptide in accordance with the presentinvention one optionally prepares an expression vector that comprises anucleic acid under the control of one or more promoters. To bring acoding sequence “under the control of” a promoter, one positions the 5′end of the translational initiation site of the reading frame generallybetween about 1 and 50 nucleotides “downstream” of (i.e., 3′ of) thechosen promoter. The “upstream” promoter stimulates transcription of theinserted DNA and promotes expression of the encoded recombinant protein.This is the meaning of “recombinant expression” in the context usedhere.

Many standard techniques are available to construct expression vectorscontaining the appropriate nucleic acids andtranscriptional/translational control sequences in order to achievepeptide expression in a variety of host-expression systems. Cell typesavailable for expression include, but are not limited to, bacteria, suchas E. coli and B. subtilis transformed with recombinant phage DNA,plasmid DNA or cosmid DNA expression vectors.

Certain examples of prokaryotic hosts are E. coli strain RR1, E. coliLE392, E. coli B, E. coli.chi. 1776 (ATCC No. 31537) as well as E. coliW3110 (F-, lambda-, prototrophic, ATCC No. 273325); bacilli such asBacillus subtilis; and other enterobacteriaceae such as Salmonellatyphimurium, Serratia marcescens, and various Pseudomonas species.

In general, plasmid vectors containing replicon and control sequencesthat are derived from species compatible with the host cell are used inconnection with these hosts. The vector ordinarily carries a replicationsite, as well as marking sequences that are capable of providingphenotypic selection in transformed cells. For example, E. coli is oftentransformed using pBR322, a plasmid derived from an E. coli species.Plasmid pBR322 contains genes for ampicillin and tetracycline resistanceand thus provides easy means for identifying transformed cells. ThepBR322 plasmid, or other microbial plasmid or phage must also contain,or be modified to contain, promoters that can be used by the microbialorganism for expression of its own proteins.

In addition, phage vectors containing replicon and control sequencesthat are compatible with the host microorganism can be used astransforming vectors in connection with these hosts. For example, thephage lambda may be utilized in making a recombinant phage vector thatcan be used to transform host cells, such as E. coli LE392.

Further useful vectors include pIN vectors and pGEX vectors, for use ingenerating glutathione S-transferase (GST) soluble fusion proteins forlater purification and separation or cleavage. Other suitable fusionproteins are those with β-galactosidase, ubiquitin, or the like.

Promoters that are most commonly used in recombinant DNA constructioninclude the β-lactamase (penicillinase), lactose and tryptophan (trp)promoter systems. While these are the most commonly used, othermicrobial promoters have been discovered and utilized, and detailsconcerning their nucleotide sequences have been published, enablingthose of skill in the art to ligate them functionally with plasmidvectors.

For expression in Saccharomyces, the plasmid YRp7, for example, iscommonly used. This plasmid contains the trp1 gene, which provides aselection marker for a mutant strain of yeast lacking the ability togrow in tryptophan, for example ATCC No. 44076 or PEP4-1. The presenceof the trp1 lesion as a characteristic of the yeast host cell genomethen provides an effective environment for detecting transformation bygrowth in the absence of tryptophan.

Suitable promoting sequences in yeast vectors include the promoters for3-phosphoglycerate kinase or other glycolytic enzymes, such as enolase,glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phosphoglucose isomerase, and glucokinase. In constructing suitableexpression plasmids, the termination sequences associated with thesegenes are also ligated into the expression vector 3′ of the sequencedesired to be expressed to provide polyadenylation of the mRNA andtermination.

Other suitable promoters, which have the additional advantage oftranscription controlled by growth conditions, include the promoterregion for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase,degradative enzymes associated with nitrogen metabolism, and theaforementioned glyceraldehyde-3-phosphate dehydrogenase, and enzymesresponsible for maltose and galactose utilization.

In addition to microorganisms, cultures of cells derived frommulticellular organisms may also be used as hosts. In principle, anysuch cell culture is operable, whether from vertebrate or invertebrateculture. In addition to mammalian cells, these include insect cellsystems infected with recombinant virus expression vectors (e.g.,baculovirus); and plant cell systems infected with recombinant virusexpression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaicvirus, TMV) or transformed with recombinant plasmid expression vectors(e.g., Ti plasmid) containing one or more coding sequences.

In a useful insect system, Autographica californica nuclear polyhedrosisvirus (AcNPV) is used as a vector to express foreign genes. The virusgrows in Spodoptera frugiperda cells. The isolated nucleic acid codingsequences are cloned into non-essential regions (for example thepolyhedron gene) of the virus and placed under control of an AcNPVpromoter (for example, the polyhedron promoter). Successful insertion ofthe coding sequences results in the inactivation of the polyhedron geneand production of non-occluded recombinant virus (i.e., virus lackingthe proteinaceous coat coded for by the polyhedron gene). Theserecombinant viruses are then used to infect Spodoptera frugiperda cellsin which the inserted gene is expressed (e.g., U.S. Pat. No. 4,215,051).

Examples of useful mammalian host cell lines are VERO and HeLa cells,Chinese hamster ovary (CHO) cell lines, W138, BHK, COS-7, 293, HepG2,NIH3T3, RIN and MDCK cell lines. In addition, a host cell may be chosenthat modulates the expression of the inserted sequences, or modifies andprocesses the gene product in the specific fashion desired. Suchmodifications (e.g., glycosylation) and processing (e.g., cleavage) ofprotein products may be important for the function of the encodedprotein.

Different host cells have characteristic and specific mechanisms for thepost-translational processing and modification of proteins. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. Expressionvectors for use in mammalian cells ordinarily include an origin ofreplication (as necessary), a promoter located in front of the gene tobe expressed, along with any necessary ribosome binding sites, RNAsplice sites, polyadenylation site, and transcriptional terminatorsequences. The origin of replication may be provided either byconstruction of the vector to include an exogenous origin, such as maybe derived from SV40 or other viral (e.g., Polyoma, Adeno, VSV, BPV)source, or may be provided by the host cell chromosomal replicationmechanism. If the vector is integrated into the host cell chromosome,the latter is often sufficient.

The promoters may be derived from the genome of mammalian cells (e.g.,metallothionein promoter) or from mammalian viruses (e.g., theadenovirus late promoter; the vaccinia virus 7.5K promoter). Further, itis also possible, and may be desirable, to utilize promoter or controlsequences normally associated with the desired gene sequence, providedsuch control sequences are compatible with the host cell systems.

A number of viral based expression systems may be utilized, for example,commonly used promoters are derived from polyoma, Adenovirus 2,cytomegalovirus and Simian Virus 40 (SV40). The early and late promotersof SV40 virus are useful because both are obtained easily from the virusas a fragment which also contains the SV40 viral origin of replication.Smaller or larger SV40 fragments may also be used, provided there isincluded the approximately 250 bp sequence extending from the HindIIIsite toward the BglI site located in the viral origin of replication.

In cases where an adenovirus is used as an expression vector, the codingsequences may be ligated to an adenovirus transcription/translationcontrol complex, e.g., the late promoter and tripartite leader sequence.This chimeric gene may then be inserted in the adenovirus genome by invitro or in vivo recombination. Insertion in a non-essential region ofthe viral genome (e.g., region E1 or E3) will result in a recombinantvirus that is viable and capable of expressing proteins in infectedhosts.

Specific initiation signals may also be required for efficienttranslation of the claimed isolated nucleic acid coding sequences. Thesesignals include the ATG initiation codon and adjacent sequences.Exogenous translational control signals, including the ATG initiationcodon, may additionally need to be provided. One of ordinary skill inthe art would readily be capable of determining this need and providingthe necessary signals. It is well known that the initiation codon mustbe in-frame (or in-phase) with the reading frame of the desired codingsequence to ensure translation of the entire insert. These exogenoustranslational control signals and initiation codons can be of a varietyof origins, both natural and synthetic. The efficiency of expression maybe enhanced by the inclusion of appropriate transcription enhancerelements or transcription terminators.

In eukaryotic expression, one will also typically desire to incorporateinto the transcriptional unit an appropriate polyadenylation site if onewas not contained within the original cloned segment. Typically, thepoly A addition site is placed about 30 to 2000 nucleotides “downstream”of the termination site of the protein at a position prior totranscription termination.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines that stably expressconstructs encoding proteins may be engineered. Rather than usingexpression vectors that contain viral origins of replication, host cellscan be transformed with vectors controlled by appropriate expressioncontrol elements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched medium, and then areswitched to a selective medium. The selectable marker in the recombinantplasmid confers resistance to the selection and allows cells to stablyintegrate the plasmid into their chromosomes and grow to form foci,which in turn can be cloned and expanded into cell lines.

A number of selection systems may be used, including, but not limited,to the herpes simplex virus thymidine kinase, hypoxanthine-guaninephosphoribosyltransferase and adenine phosphoribosyltransferase genes,in tk⁻, hgprt⁻ or aprt⁻ cells, respectively. Also, antimetaboliteresistance can be used as the basis of selection for dhfr, which confersresistance to methotrexate; gpt, which confers resistance tomycophenolic acid; neo, which confers resistance to the aminoglycosideG-418; and hygro, which confers resistance to hygromycin. It isappreciated that numerous other selection systems are known in the artthat are similarly operable in the present invention.

It is contemplated that the isolated nucleic acids of the disclosure maybe “overexpressed”, i.e., expressed in increased levels relative to itsnatural expression in cells of its indigenous organism, or even relativeto the expression of other proteins in the recombinant host cell. Suchoverexpression may be assessed by a variety of methods, includingradio-labeling and/or protein purification. However, simple and directmethods are preferred, for example, those involving SDS/PAGE and proteinstaining or immunoblotting, followed by quantitative analyses, such asdensitometric scanning of the resultant gel or blot. A specific increasein the level of the recombinant protein or peptide in comparison to thelevel in natural human cells is indicative of overexpression, as is arelative abundance of the specific protein in relation to the otherproteins produced by the host cell and, e.g., visible on a gel.

A nucleic acid of this invention can be in a cell, which can be a cellexpressing the nucleic acid whereby a peptide of this invention isproduced in the cell. In addition, the vector of this invention can bein a cell, which can be a cell expressing the nucleic acid of the vectorwhereby a peptide of this invention is produced in the cell. It is alsocontemplated that the nucleic acids and/or vectors of this invention canbe present in a host animal (e.g., a transgenic animal) which expressesthe nucleic acids of this invention and produces the peptides of thisinvention.

The nucleic acid encoding the peptides of this invention can be anynucleic acid that functionally encodes the peptides of this invention.To functionally encode the peptides (i.e., allow the nucleic acids to beexpressed), the nucleic acid of this invention can include, for example,expression control sequences, such as an origin of replication, apromoter, an enhancer and necessary information processing sites, suchas ribosome binding sites, RNA splice sites, polyadenylation sites andtranscriptional terminator sequences.

Expression control sequences include promoters derived frommetallothionine genes, actin genes, immunoglobulin genes, CMV, SV40,adenovirus, bovine papilloma virus, etc. A nucleic acid encoding aselected peptide can readily be determined based upon the genetic codefor the amino acid sequence of the selected peptide and many nucleicacids will encode any selected peptide. Modifications in the nucleicacid sequence encoding the peptide are also contemplated. Modificationsthat can be useful are modifications to the sequences controllingexpression of the peptide to make production of the peptide inducible orrepressible as controlled by the appropriate inducer or repressor. Suchmethods are standard in the art. The nucleic acid of this invention canbe generated by means standard in the art, such as by recombinantnucleic acid techniques and by synthetic nucleic acid synthesis or invitro enzymatic synthesis.

An inventive peptide of the present invention is optionally modified toincrease its immunogenicity. In a non-limiting example, the antigen iscoupled to chemical compounds or immunogenic carriers, provided that thecoupling does not interfere with the desired biological activity ofeither the antigen or the carrier. For a review of some generalconsiderations in coupling strategies, see Antibodies, A LaboratoryManual, Cold Spring Harbor Laboratory, ed. E. Harlow and D. Lane (1988).Useful immunogenic carriers known in the art, include, withoutlimitation, keyhole limpet hemocyanin (KLH); bovine serum albumin (BSA),ovalbumin, PPD (purified protein derivative of tuberculin); red bloodcells; tetanus toxoid; cholera toxoid; agarose beads; activated carbon;or bentonite. Useful chemical compounds for coupling include, withoutlimitation, dinitrophenol groups and arsonilic acid.

The inventive polypeptide may also be modified by other techniques,illustratively including denaturation with heat and/or SDS.

In another aspect, the invention provides a multi-component vaccine.Optionally, a multi-component vaccine contains more than one immunogen.An inventive vaccine may contain 2, 3, 4, 5, 6, 7, 8, 9, 10, or moreimmunogens in a single vaccine. Optionally, a first immunogen is apeptide corresponding to amino acid position 301 to amino acid position330 of SEQ ID NO: 1, a fragment thereof, or an analogue thereof. It isappreciated that any of the aforementioned modifications, mutations, oralterations stated herein or otherwise known in the art are operable asto the inventive immunogens of the present invention.

Optionally, an inventive vaccine contains an adjuvant. Suitableadjuvants illustratively include dimethyl dioctadecyl-ammonium bromide(DDA); monophosphoryl lipid A (MPL); LTK63, lipophilic quaternaryammonium salt-DDA, DDA-MPL, aluminum salts, aluminum hydroxide, aluminumphosphate, potassium aluminum phosphate, Montanide ISA-51, ISA-720,microparticles, immunostimulatory complexes, liposomes, virosomes,virus-like particles, CpG oligonucleotides, cholera toxin, heat-labiletoxin from E. coli, lipoproteins, dendritic cells, IL-12, GM-CSF,nanoparticles illustratively including calcium phosphate nanoparticles,combination of soybean oil, emulsifying agents, and ethanol to form ananoemulsion; AS04, ZADAXIN, or combinations thereof.

The peptide vaccine is optionally delivered as naked polypeptide, inaqueous solution, in an emulsion, or in other suitable deliverycomposition. In some embodiments, the invention is delivered as avaccine or as a vaccine component of a pharmaceutical package.Optionally, a peptide (or multiple peptides) is present in an emulsionincluding one or more emulsification agents. In some embodiments, amulticomponent vaccine is emulsified. In some embodiments a singlesubunit vaccine is emulsified. Suitable emulsification agentsillustratively include supramolecular biovectors (SMBV), nanoparticlessuch as described by Major, M, et al, Biochim. Biophys. Acta, 1997;1327:32-40, De Migel, I, et al, Pharm. Res., 2000; 17:817-824, U.S. Pat.Nos. 6,017,513, 7,097,849, 7,041,705, 6,979,456, 6,846,917, 6,663,861,6,544,646, 6,541,030, 6,368,602, Castignolles, N., et el, Vaccine, 1996;14:1353-1360, Prieur, E., et al, Vaccine, 1996; 14:511-520, Baudner B,et al, Infect Immun, 2002; 70:4785-4790; Liposomes such as described byEl Guink et al., Vaccine, 1989; 7:147-151, and in U.S. Pat. No.4,196,191; or other agents known in the art. Agents suitable for use aregenerally available from Sigma-Aldrich, St. Louis, Mo. Theemulsification agent is optionally a dimethyl dioctadecyl-ammoniumbromide. Optionally the adjuvant is monophosphoryl lipid A.

Suitable pharmaceutically acceptable carriers facilitate administrationof the immunogens are physiologically inert and/or nonharmful. Carriersmay be selected by one of skill in the art. Exemplary carriers includesterile water or saline, lactose, sucrose, calcium phosphate, gelatin,dextran, agar, pectin, peanut oil, olive oil, sesame oil, and water.Additionally, the carrier or diluent may include a time delay material,such as glycerol monostearate or glycerol distearate alone or with awax. In addition, slow release polymer formulations can be used.

Optionally, the inventive composition may also contain conventionalpharmaceutical ingredients, such as preservatives, or chemicalstabilizers. Suitable ingredients operable herein include, for example,casamino acids, sucrose, gelatin, phenol red, N-Z amine, monopotassiumdiphosphate, lactose, lactalbumin hydrolysate, and dried milk.

Immunological compositions and other pharmaceutical compositionscontaining the peptide(s) described herein are included within the scopeof the present invention. One or more of these compositions can beformulated and packaged, alone or in combination, using methods andmaterials known to those skilled in the art for vaccines. Theimmunological response may be therapeutic or prophylactic and mayprovide antibody immunity or cellular immunity such as that produced byT lymphocytes such as cytotoxic T lymphocytes or CD4⁺ T lymphocytes.

The inventive vaccines may be administered with an adjuvant. Optionally,an adjuvant is alum (aluminum phosphate or aluminum hydroxide).Chemically defined preparations such as muramyl dipeptide,monophosphoryl lipid A, phospholipid conjugates, encapsulation of theconjugate within a proteoliposome, and encapsulation of the protein inlipid vesicles are also operable with the present invention.

Suitable methods of administration include, but are not limited tointramuscular, intravenous, intranasal, mucosal, oral, parenteral,intravaginal, transdermal, via aerosol delivery or by any route thatproduces the desired biological effect or immune response.

A vaccine of the invention is optionally packaged in a single dosage forimmunization by parenteral (i.e., intramuscular, intradermal orsubcutaneous) administration or nasopharyngeal (i.e., intranasal)administration. The vaccine is optionally delivered by inhalation. Thevaccine is optionally combined with a pharmaceutically acceptablecarrier to facilitate administration. The carrier is usually water or abuffered saline, with or without a preservative. The vaccine may belyophilized for resuspension at the time of administration or insolution.

Optional microencapsulation of the inventive vaccine will also provide acontrolled release. A number of factors contribute to the selection of aparticular polymer for microencapsulation. The reproducibility ofpolymer synthesis and the microencapsulation process, the cost of themicroencapsulation materials and process, the toxicological profile, therequirements for variable release kinetics and the physicochemicalcompatibility of the polymer and the antigens are all factors that maybe considered. Examples of useful polymers illustratively includepolycarbonates, polyesters, polyurethanes, polyorthoesters polyamides,poly(d,l-lactide-co-glycolide) (PLGA) and other biodegradable polymers.

The inventive vaccine may additionally contain stabilizers such asthimerosal (ethyl(2-mercaptobenzoate-S)mercury sodium salt) (SigmaChemical Company, St. Louis, Mo.) or physiologically acceptablepreservatives.

Additional, a human or other animal may be treated for anthrax infectionby administering an effective amount of an immunogen of the invention.An “effective amount” is optionally between about 0.05 to about 1000μg/mL of an immunogen. A suitable dosage may be about 1.0 mL of such aneffective amount. Such a composition may be administered 1-3 times perday over a 1 day to 12 week period. However, suitable dosage adjustmentsmay be made by the attending physician or veterinarian depending uponthe age, sex, weight and general health of the subject. Such acomposition is optionally administered parenterally, optionallyintramuscularly or subcutaneously. However, it may also be formulated tobe administered by any other suitable route, including orally ortopically.

Embodiments of inventive compositions and methods are illustrated in thefollowing examples. These examples are provided for illustrativepurposes and are not considered limitations on the scope of inventions.

EXAMPLES Example 1 Synthesis of Peptides

Fmoc synthesis is used to prepare 37 N-terminally biotinylated peptidesof 30 amino acid (AA) residues each, overlapping by 10 AA representingsequences of PA (SEQ ID NO: 1). The peptide representing the C-terminusof SEQ ID NO: 1 is made as the free acid. All sequences are Fmocsynthesized as C-terminal amides, HPLC-purified, and prepared astrifluoracetic acid salts.

Example 2 Screening of Sera from Rhesus Macaques, Rabbits and HumanSubjects Immunized with a Vaccine Including PA Sequences for AcquiredImmunity

AVA vaccine (BIOTHRAX), and rPA vaccine (PREVITHRX) are obtained fromEmergent Biosolutions, Rockville, Md. New Zealand white rabbits andrhesus macaques are vaccinated by intramuscular injection at 0, 4, and 8weeks with 0.5 ml of either AVA or rPA vaccine in either undiluted formor a 1:5 dilution of the normal human dose. Rabbits are vaccinated witheither an undiluted dose or an 1:10 or 1:20 dilution of the normal humandose. All dilutions are in saline. As a control, animals are vaccinatedwith an equal volume of saline at the same intervals.

Ten human subjects are vaccinated with AVA at the recommended dose andschedule of administration following the regimen at the time of thestudy with administration at 0-2-4 weeks and 6-12-30-42 months withannual boosters.

Whole blood is obtained from human and macaque subjects by venipuncture.Whole blood from rabbits is obtained by cardiac puncture. All blood iscollected into tubes in the absence of an anticoagulant. Tubes areallowed to incubate on the bench top for 45 minutes to induce clotformation. The resulting serum is separated by centrifugation at 2000×gfor 15 minutes and aspirated as the supernatant. All samples are eitherassayed immediately or stored at −80° C. until assay.

A 96-well assay microplate is coated with streptavidin (2 μg/ml in eachwell). The biotinylated peptides of Example 1 are bound to individualwells of the plate. Appropriate controls are assayed simultaneously onthe same assay plate in independent wells. Test serum is diluted 1:41 inphosphate buffered saline (pH 7.4) as sample diluent. Each diluted serumsample is then divided among the test wells with each individual peptidescreened in duplicate on the same plate. 100 μl of diluted serum isplaced in each appropriate well and incubated at room temperature for 30minutes. The liquid is removed from each well by aspiration followed bythree washes with phosphate buffered saline. The final liquid isaspirated and the wells are tapped dry. 100 μl of species specificanti-IgG conjugated to horseradish peroxidase is added to each well andallowed to incubate for 10 minutes at room temperature. The wells arethen washed three times with phosphate buffered saline and tapped dry.Each well is then incubated in the presence of 100 μl of TMB-substratesolution for 10 minutes followed by the addition of 100 μl of 0.16 Msulfuric acid as a stop solution. The level of product formed isdetermined spectrophotometrically by determination of optical density at450 nm.

The reactivity of serum from three different pools of vaccinated Rhesusmacaques are illustrated in FIG. 1. At least one peptide representingamino acid sequence from each of the four domains of PA is a target forPA specific IgG in Rhesus macaques. Particularly strong reactivity isobserved in domain 2 between amino acids 301 and 330 as well as invarious locations in domain 4. Macaque AVR817 is a negative control anddemonstrates baseline activity throughout PA.

Interestingly, similar profiles of reactivity are observed in sera fromrabbits and humans vaccinated with AVA. FIG. 2 demonstrates strongreactivity in domain 2 for peptides corresponding to residues 301-330 ofmature PA. Rabbits also show strong reactivity in a range of domain 3regions and domain 4 regions. Humans show the most restricted reactivityof the three species, but the overall regions of reactivity are similarwith high reactivity in domain 2 at amino acids 301-330 and in domain 4.

The seroreactivity is independent on the type of vaccine used tovaccinate both humans and Rhesus macaques. FIG. 4 illustrates thereactivity in sera from Rhesus macaques vaccinated either with AVA orrPA vaccines and show highly correlative reactivity. Additionally, theaddition of four administrations in macaques to a total of 7 does notalter the reactivity indicating that full immunity is observed afterthree administrations. (FIG. 5)

Overall, vaccination with rPA or AVA vaccines produces a robust immuneresponse with the generation of antibodies to several regions of PAencompassing domains 1-4. Little to no reactivity is observed at theN-terminus of PA (residues 1-167) in all three species.

Example 3 Production of Peptide Vaccines

Fmoc synthesis is used to prepare 11 peptide vaccines. The peptidevaccines have the following sequences describing the amino acidnumbering of SEQ ID NO: 1: the calcium ion chelating residues in the 1α₁and 1β₁₃ strands (AA181-210) (DGIPDSLEVEGYTVDVKNKRTFLSPWISNI (SEQ ID NO:4)); 1β₁₃, 1α₂, 1β₁₄ (AA201-230) (TFLSPWISNIHEKKGLTKYKSSPEKWSTAS (SEQ IDNO: 5)); 1α₃ and 1α₄ (AA 221-250) (SSPEKWSTASDPYSDFEKVTGRIDKNVSPE (SEQID NO: 6)); and 1α₄ and 2β₁ (AA241-270) (GRIDKNVSPEARHPLVAAYPIVHVDMENIISEQ ID NO: 7)); the chymotrypsin sensitive loop 2β₂-2β₃ (AA 301-330)(SEVHGNAEVHASFFDIGGSVSAGFSNSNSS (SEQ ID NO: 3)); 2β₃ and 2α₁ (AA321-350) (SAGFSNSNSSTVAIDHSLSLAGERTWAETM (SEQ ID NO: 8)); 2α₁, 2β₄ and2β₅ (AA341-370) (AGERTWAETMGLNTADTARLNANIRYVNTG (SEQ ID NO: 9); 2β₆ and2β₇ (AA361-390) (NANIRYVNTGTAPIYNVLPTTSLVLGKNQT (SEQ ID NO: 10); 2β₁₀,2β₁₁, 2α₂ and 2β₁₂ (AA421-450) (LNAQDDFSSTPITMNYNQFLELEKTKQLRL (SEQ IDNO: 11)); 2β₁₃ in domain 2, 3α₃, 3α₄ (AA 561-590)(NIKNQLAELNVTNIYTVLDKIKLNAKMNIL (SEQ ID NO: 12)); and 3β₇, 3β₈(AA581-610) (IKLNAKMNILIRDKRFHYDRNNIAVGADES (SEQ ID NO: 13)). Controlmice are immunized with a scrambled peptide of 30 amino acids in length.All sequences are Fmoc synthesized as C-terminal amides, HPLC-purified,and prepared as trifluoracetic acid salts.

The immunization studies in mice are performed in accordance withfederal and institutional guidelines. Seven groups of five female 6- to7-week-old BALB/c mice (Charles River) are immunized subcutaneously(s.c.; 1 μg peptide) at two sites (100 μl per site) on day 0. Boosterdoses are administered on day 14, day 28, and day 140. Blood is drawnfrom the retro-orbital plexus 15 days after the third and fourth doses(i.e., on days 43 and 155 of post-initial immunization). The bloodsamples are allowed to stay undisturbed for 2 h at room temperature,stored at 4° C. overnight, and centrifuged at 3,000 rpm for 10 min toextract the serum for analysis for the presence of antibodies to PA.

For detection of PA specific antibodies, 96-well microtiter ELISA platesare coated with 100 μl/well of PA standard at a concentration of 2.0μg/ml in PBS, pH 7.4. The plates are stored overnight at 4° C. The serumsamples from the mouse are serially diluted (1:100 to 1:640,000). Platesare incubated with 100 μl of diluted serum samples for 1 h at 37° C.followed by washing with PBS-Tween. The plates are then incubated for 1h at 37° C. with 100 μl of HRP-conjugated goat anti-mouse IgG (1:5,000dilution of 1-mg/ml stock). TMB is used as the substrate, and thereaction was stopped by adding 50 μl of 2 M sulfuric acid. The platesare read on a plate reader (Dynex Technologies) at 450 nm. Titer valuesare calculated using a cutoff value equal to an absorbance difference of0.5 between immunized and unimmunized mice.

Each of the immunogens elicits the production of antibodies in mice withthe exception of the control peptide that is at background levels.

The same immunogens are used to vaccinate humans. Each immunogen isadministered subcutaneously (s.c.) in 8 doses at an immunization amountof 5 μg. Sera from each subject is then assayed for the presence ofantibodies to each peptide as described above. Each of the immunogensproduces antibodies in human serum.

Serum samples are collected at the time of each human subjectadministration. A profile of the presence of antibodies to PA and thelevel of antibodies in each subject's serum is determined by ELISA.Human subjects show little antibody production after the first andsecond administrations indicating that at least one additional boosterimmunization is required to develop required immunity. The levels ofantibodies are sufficiently present after three administrations.Subsequent administrations do not significantly increase the level ofserum antibodies. Thus, three administrations is sufficient to conferimmunity in most human subjects. Two subjects do not show the presenceof robust anti-PA antibody levels after three administrations. Thesesubjects are determined to require at least one additionaladministration. After a fourth administration the level of PA-antibodiesis greater than background indicating the presence of acquired immunity.

Example 4 Anthrax Toxin Neutralization

Sera from immunized mice as in Example 3 are tested for neutralizationin a macrophage cytotoxicity assay essentially as described by Koya, V.et al, Infection and Immunity, 2005, 73:8266-8274. Briefly, serum fromeach mouse is diluted directly into 96-well LTx plates with LTx (PA plusLF) previously added at 50 ng/ml in Dulbecco's modified Eagle's medium(100 μl/well, except 150 μl in first well). Serum is added starting at1:150 and proceeding in 3.14-fold dilutions and incubated for 30minutes. Each serum is tested in triplicate. 90 μl of the serum/LTxmixture is moved to a second 96-well plate containing RAW264.7 cellsgrown to 90% confluence and incubated for 5 h at 37° C. Cell death isassessed by addition of MTT[3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide] (Sigma,St. Louis, Mo.) at a final concentration of 0.5 mg/ml, incubated for 40minutes, and the blue pigment produced by viable cells is dissolved byaspirating the medium and adding 50 μl/well of a mixture containing 0.5%(wt/vol) SDS and 25 mM HCl in 90% (vol/vol) isopropanol and shaking theplates for 5 min prior to reading at 570 nm using a microplate reader.

Sera from each mouse vaccinated with a peptide immunogen producesantibodies that neutralize toxin.

Methods involving conventional biological techniques are describedherein. Such techniques are generally known in the art and are describedin detail in methodology treatises such as Molecular Cloning: ALaboratory Manual, 3rd ed., vol. 1-3, ed. Sambrook et al., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 2001; CurrentProtocols in Molecular Biology, ed. Ausubel et al., Greene Publishingand Wiley-Interscience, New York, 1992 (with periodic updates); andShort Protocols in Molecular Biology, ed. Ausubel et al., 52 ed.,Wiley-Interscience, New York, 2002. Immunological methods (e.g.,preparation of antigen-specific antibodies, immunoprecipitation, andimmunoblotting) are described, e.g., in Current Protocols in Immunology,ed. Coligan et al., John Wiley & Sons, New York, 1991; and Methods ofImmunological Analysis, ed. Masseyeff et al., John Wiley & Sons, NewYork, 1992.

Methods of producing and screening antibodies are illustratively foundin Monoclonal Antibodies: Methods and Protocols, Albitar, M, ed., HumanaPress, 2010 (ISBN 1617376469); and Antibodies: A Laboratory Manual,Harlos, E, and Lane, D. eds., Cold Spring Harbor Laboratory Press, 1988(ISBN-10: 0879693142).

Additional protocols such as PCR Protocols can be found in A Guide toMethods and Applications Academic Press, NY. Methods for proteinpurification include such methods as ammonium sulfate precipitation,column chromatography, electrophoresis, centrifugation, crystallization,and others. See, e.g., Ausubel, et al. (1987 and periodic supplements);Deutscher (1990) “Guide to Protein Purification,” Methods in Enzymologyvol. 182, and other volumes in this series; Current Protocols in ProteinScience, John Wiley and Sons, New York, N.Y.; and manufacturer'sliterature on use of protein purification products known to those ofskill in the art.

Various modifications of the present invention, in addition to thoseshown and described herein, will be apparent to those skilled in the artof the above description. Such modifications are also intended to fallwithin the scope of the appended claims.

It is appreciated that all reagents are obtainable by sources known inthe art unless otherwise specified. Methods of nucleotide amplification,cell transfection, and protein expression and purification are similarlywithin the level of skill in the art.

Patents and publications mentioned in the specification are indicativeof the levels of those skilled in the art to which the inventionpertains. These patents and publications are incorporated herein byreference to the same extent as if each individual application orpublication was specifically and individually incorporated herein byreference.

The foregoing description is illustrative of particular embodiments ofthe invention, but is not meant to be a limitation upon the practicethereof. The following claims, including all equivalents thereof, areintended to define the scope of the invention.

The invention claimed is:
 1. A Bacillus anthracis vaccine comprising anisolated immunogen consisting of 10 to 30 amino acids in the sequence ofat least one of the amino acid regions 181-210, 201-230, 221-250,241-270, 321-350, 341-370, 361-390, 421-450, 561-590, or 581-610 of SEQID NO: 1, or a combination thereof.
 2. The vaccine of claim 1 whereinsaid vaccine comprises multiple amino acid regions of Bacillus anthracisprotective antigen.
 3. The vaccine of claim 1 wherein said immunogen isrecombinant.
 4. The vaccine of claim 1 wherein said immunogen furthercomprises a tag suitable for purification.
 5. The Bacillus anthracisvaccine of claim 1 wherein the immunogen consists of 30 amino acids. 6.A Bacillus anthracis vaccine comprising a plurality of isolatedimmunogens, said plurality comprising two or more of: a first isolatedimmunogen consisting of an amino acid sequence of 181-210 of SEQ ID NO:1; a second isolated immunogen consisting of an amino acid sequence of201-230 of SEQ ID NO: 1; a third isolated immunogen consisting of anamino acid sequence of 221-250 of SEQ ID NO: 1; a fourth isolatedimmunogen consisting of an amino acid sequence of 241-270 of SEQ ID NO:1; a fifth isolated immunogen consisting of an amino acid sequence of321-350 of SEQ ID NO: 1; a sixth isolated immunogen consisting of anamino acid sequence of 341-370 of SEQ ID NO: 1; a seventh isolatedimmunogen consisting of an amino acid sequence of 361-390 of SEQ ID NO:1; an eighth isolated immunogen consisting of an amino acid sequence of421-450 of SEQ ID NO: 1; a ninth isolated immunogen consisting of anamino acid sequence of 561-590 of SEQ ID NO: 1; and a tenth isolatedimmunogen consisting of an amino acid sequence of 581-610 of SEQ IDNO:
 1. 7. The vaccine of claim 6 comprising said first isolatedimmunogen, said second isolated immunogen, said third isolatedimmunogen, said fifth isolated immunogen, said sixth isolated immunogen,said seventh isolated immunogen, said eighth isolated immunogen, saidninth isolated immunogen, and said tenth isolated immunogen.
 8. ABacillus anthracis vaccine comprising: an isolated immunogen consistingof amino acids in the sequence of at least one of the amino acid regions181-210, 201-230, 221-250, 241-270, 321-350, 341-370, 361-390, 421-450,561-590, or 581-610 of SEQ ID NO: 1, or a combination thereof, whereinthe immunogen consists of 10 to 30 amino acids; and an adjuvant.